Deoxyribonuclease activities in Lactobacillus delbrueckii

DNase activity was examined in the extracellular and subcellular fractions of six non-transformable strains belonging to Lactobacillus delbrueckii subsp. lactis (L. lactis) and Lactobacillus delbrueckii subsp. bulgaricus (L. bulgaricus) and compared with the activity present in Lactobacillus johnsonii NCK 65, a transformable strain of Lactobacillus. In the extracellular fraction of the L. delbrueckii strains, a common protein band of 36 kDa was detected, while a band of 29 kDa was found in the same fraction of L. johnsonii. No nuclease activity was detected in the cytoplasmic fraction of this strain, indicating that the localization of the DNase activity could be a key factor in the uptake of foreign DNA.     

Enzymatic activities in cell fractions of mycoplasmalike organisms purified from aster yellows-infected plants.

Mycoplasmalike organisms (MLOs), purified from aster yellows-infected plants were osmotically lysed, and the membranes were separated from the cytoplasmic fraction through differential centrifugation. Electron microscopic examinations of sections of the purified MLOs and the isolated membranes showed pleomorphic bodies and unit membranous empty vesicles, respectively. Cell fractions were tested for NADH oxidase, NADPH oxidase, ATPase, RNase, DNase, and p-nitrophenyl phosphatase activity. NADH oxidase and ATPase were confined to the membrane fraction and NADPH oxidase to the cytoplasmic fraction of the MLOs. para-Nitrophenyl phosphatase, RNase, and DNase activities were detected in both membrane and cytoplasmic fractions, but p-nitrophenyl phosphatase and RNase appeared to be associated with membranes and DNase with the cytoplasmic fraction. Glucose-6-phosphate dehydrogenase was found in the cytoplasmic fraction of the MLO cells. Our findings on the distribution of enzymes in MLO cells and cell fractions are the first basic documentation on nonhelical, nonculturable microbes parasitic to plants.     

Role of DNase in recovery of plasmid DNA from Clostridium perfringens.

Recovery of plasmid DNA from Clostridium perfringens 10543A and 3626B cleared lysates was significantly improved by the addition of 0.2% (vol/vol) diethylpyrocarbonate (DEP) before protoplast disruption in the cleared lysate protocol. Three previously undetected, large-molecular-mass plasmids (45.2, 51.9, and 68.2 megadaltons) were isolated from modified DEP-treated cleared lysates of C. perfringens 3626B. Two plasmids (9.4 and 30 megadaltons) were recovered from C. perfringens 10543A modified DEP-treated cleared lysates which previously required dye-buoyant density gradient centrifugation for visualization on agarose gels. Unsuccessful attempts to isolate plasmid DNA from Brij 58 cleared lysates of extracellular DNase-negative mutants of C. perfringens suggested the deleterious DNase activity was not extracellular. Cellular localization studies indicated that the cell wall-compartmentalized cell fraction contained 72.2% of the total DNase activity, whereas the extracellular and intracellular fractions demonstrated much less (26.8 and 1.0%, respectively). Cleared lysates prepared with DEP demonstrated much less DNase activity than cleared lysates prepared without DEP. The variable and irreproducible recovery of plasmid DNA from C. perfringens cleared lysates was attributed to cell wall-compartmentalized DNase.     

Role of DNase in recovery of plasmid DNA from Clostridium perfringens

Recovery of plasmid DNA from Clostridium perfringens 10543A and 3626B cleared lysates was significantly improved by the addition of 0.2% (vol/vol) diethylpyrocarbonate (DEP) before protoplast disruption in the cleared lysate protocol. Three previously undetected, large-molecular-mass plasmids (45.2, 51.9, and 68.2 megadaltons) were isolated from modified DEP-treated cleared lysates of C. perfringens 3626B. Two plasmids (9.4 and 30 megadaltons) were recovered from C. perfringens 10543A modified DEP-treated cleared lysates which previously required dye-buoyant density gradient centrifugation for visualization on agarose gels. Unsuccessful attempts to isolate plasmid DNA from Brij 58 cleared lysates of extracellular DNase-negative mutants of C. perfringens suggested the deleterious DNase activity was not extracellular. Cellular localization studies indicated that the cell wall-compartmentalized cell fraction contained 72.2% of the total DNase activity, whereas the extracellular and intracellular fractions demonstrated much less (26.8 and 1.0%, respectively). Cleared lysates prepared with DEP demonstrated much less DNase activity than cleared lysates prepared without DEP. The variable and irreproducible recovery of plasmid DNA from C. perfringens cleared lysates was attributed to cell wall-compartmentalized DNase.     

Mitochondrial DNA polymerase, deoxyribonuclease and ribonuclease H activities from brain of chick embryo

R-DNA polymerase, D-DNA polymerase, DNase and RNase H activities in mitochondria from chick embryonic brain were studied by ion-exchange chromatography. Two main fractions were separated according to their chromatographic behaviour: a fraction M Ib which is eluted with the washing buffer from two successive DEAE-cellulose columns and a fraction M IV which is eluted at 400 mM KC1 from a phosphocellulose column. Although the two fractions contain both the DNA polymerase and the degrading activities, all the specific activities are higher in fraction M IV than in fraction M Ib. Heat inactivation experiments have shown that R-DNA polymerase is inactivated in both fractions, whereas RNase H and DNase are not affected. Thus, degrading activities and R-DNA polymerase activity seem to be catalyzed by different molecular entities. However the fact that in most cases these activities co-chromatograph suggests that the corresponding molecules form rather stable complexes.     

Effect of antigenic stimulation on acid deoxyribonuclease activity in lymphocytes: I. Antibody-induced response

The effect of antigen-induced stimulation on acid deoxyribonuclease (DNase) activity in BALB/c mouse lymphoid cells was determined. Increase in acid DNase activity was found in intact spleen cell populations of mice from the second or fourth day after immunization with pneumococcal polysaccharide type III and from the fourth day after immunization with SRBC. DNase determinations performed with spleen cell fractions prepared from SRBC-immunized mice, showed that the rise in the enzyme activity was confined to the fraction containing the antibody-forming cells. The DNase activity was also increased in spleen cell cultures, stimulated with SRBC in vitro. Rise in the activity of this enzyme was also observed in peritoneal cell populations taken from SRBC-immunized mice. This change was maximal on the second day after immunization, when no appreciable increase in DNase activity of spleen cells was yet detected. The results obtained suggest, that acid DNase is an enzyme involved in the proliferative/maturation response to antigenic stimulation. It is a consequence of antigenic stimulation rather than being involved in the process of afferent stimulation.     

DNase-Resistant DNA in the Extracellular and Cell Wall-Associated Fractions of Frankia Strains R43 and CcI3

DNases were shown to be present in the extracellular fraction of Frankia strains R43 and CcI3. In spite of this, DNA was found in both the extracellular and cell wall fractions of these strains, and it was shown that extracellular DNA was resistant to the DNases secreted into the culture medium of both Frankia strains. Furthermore, Southern blot analysis under high stringency conditions revealed the chromosomal origin of the cell wall-adsorbed DNA (CW-DNA). Mobility gel band shift assays suggested that the extracellular DNA and the CW-DNA are engaged in complexes with other molecules, most likely proteins, which are probably responsible for the enzymatic resistance observed against extracellular DNase activities. In addition, it was shown that lysis of a small proportion of the cells in the exponential growth phase may account for the DNA being released into the supernatant and adsorbed to the cell wall. Received: 21 July 2000 / Accepted: 21 August 2000     

Bioactivity studies of Huh-7 cells derived human epidermal growth factor expressed in Pichia pastoris

Previously, we have reported cloning of human epidermal growth factor gene from Huh-7 cells and its extracellular expression in Pichia pastoris. The presented work is a detailed report regarding molecular characterization of Huh-7 cells-derived hEGF expressed in Pichia pastoris with special reference to its glycosylation profiling and bioactivity studies. Densitometric scanning of SDS-PAGE separated extracellular proteins from hEGF recombinant Pichia pastoris strain indicated that about 84% of the extracellular proteins were glycosylated. Size exclusion chromatography using Superdex 75 prep grade column was successfully utilized to separate fractions containing glycosylated and non-glycosylated extracellular proteins. In dot blot assay, hEGF was detected in both glycosylated and non-glycosylated fractions. Bioactivity assays revealed that both glycosylated and non-glycosylated fractions were bioactive as determined by cell viability assay. It was also observed that hEGF present in non-glycosylated fraction was relatively more bioactive than hEGF present in glycosylated fraction.     

Export and intercellular transfer of DNA via membrane blebs of Neisseria gonorrhoeae.

Naturally elaborated membrane bleb material is frequently observed in cultures of Neisseria gonorrhoeae. This material was purified and analyzed for protein, lipopolysaccharide, and nucleic acid content. The electrophoretic protein profiles of two bleb-rich fractions, called BI and BII, were distinct, with only BII containing lipopolysaccharide and outer membrane proteins I and III. Both fractions contained RNA, circular DNA, and linear DNA. Exogenous pancreatic DNase I appeared to hydrolyze all bleb-associated DNA in fraction BI and the linear DNA in fraction BII. The circular DNA molecules associated with fraction BII resisted digestion. Electron microscopy of the bleb fractions verified their DNA content. Fixing blebs with glutaraldehyde before mounting them for microscopy prevented release of internal DNA. Such fixation produced little change in the micrographs of BI; however, only traces of DNA were observed in fixed BII preparations. Incubation of wild-type gonococci in mixtures of DNase and blebs purified from antibiotic-resistant strains resulted in efficient exchange of penicillinase-specifying R plasmids. Recipients incorporated plasmids independently of endogenous and exogenous chromosomal streptomycin resistance markers. These in vitro results suggest that bleb formation by N. gonorrhoeae may serve to transfer plasmids intercellularly in vivo, perhaps constituting a previously unexplored genetic exchange mechanism in these bacteria.     

The cytosolic, cell surface and extracellular proteomes of the biotechnologically important soil bacterium Corynebacterium efficiens YS-314 in comparison to those of Corynebacterium glutamicum ATCC 13032

Reference maps of the cytosolic, cell surface and extracellular proteome fractions of the amino acid-producing soil bacterium Corynebacterium efficiens YS-314 were established. The analysis window covers a pI range from 3 to 7 along with a molecular mass range from 10 to 130 kDa. After second-dimensional separation on SDS-PAGE and Coomassie staining, computational analysis detected 635 protein spots in the cytosolic proteome fraction, whereas 76 and 102 spots were detected in the cell surface and extracellular proteomes, respectively. By means of MALDI-TOF-MS and tryptic peptide mass fingerprinting, 164 cytosolic proteins, 49 proteins of the cell surface and 89 extracellular protein spots were identified, representing in total 177 different proteins. Additionally, reference maps of the three cellular proteome fractions of the close phylogenetic relative Corynebacterium glutamicum ATCC 13032 were generated and used for comparative proteomics. Classification according to the Clusters of Orthologous Groups of proteins scheme and abundance analysis of the identified proteins revealed species-specific differences. The high abundance of molecular chaperones and amino acid biosynthesis enzymes in C. efficiens points to environmental adaptations of this recently discovered amino acid-producing bacterium.     

Extracellular DNase activity of Cryptococcus neoformans and Cryptococcus gattii

Extracellular DNase activity was studied in 73 strains of Cryptococcus neoformans and 12 strains of Cryptococcus gattii. DNase activity was measured by DNase agar clearance with and without Methyl Green. All strains tested showed extracellular DNase activity and no significant difference was found betweenC. neoformans and C. gattii strains. DNase production was higher in strains from clinical origin (average radius of 6.2 mm) than among environmental strains (average radius of 2.9 mm). The extracellular enzyme may be detected by DNA substrate PAGE assays and its molecular weight was estimated at 31 kD. These results suggest that extracellular DNase could be considered as a virulence factor involved in C. neoformans–C. gattii species complex pathogenicity.     

Electrophoretic patterns of extracellular deoxyribonuclease (DNase) and their correlation with T-type in group A streptococci

Molecular heterogeneity of the extracellular deoxyribonuclease (DNase) in group A streptococci was demonstrated in 42 clinical isolates. Although polyacrylamide gel electrophoretic patterns of the extracellular DNase of all the isolates were heterogeneous, they could be divided into five main patterns with respect to the presence or absence of three DNase components including DNase B. By comparing the electrophoretic patterns of DNase in all the isolates with their T-types, we found that the patterns were quite characteristic for their T-types, especially in the prevalent T-types 12 and 1, and that the isolates of T-types 12 and 1 produced DNase B as their major extracellular DNase. Relative DNase B activity in the total extracellular DNase activity of group A, B, and G isolates was determined by the rapid method of neutralization with anti-DNase B antibody. The results showed neutralization of DNase activity in all the isolates of group A streptococci, largely corresponding to their T-types, but not of the isolates of groups B and G. These results indicate that the electrophoretic patterns of the extracellular DNase of group A streptococci are closely correlated with their T-types, suggesting the physicochemical taxonomic value of these properties.     

DNA-protein interactions and chromosome banding

Pancreatic DNase I was used as a probe to study DNA-protein interactions in condensed and extended chromatin fractions isolated from Chinese hamster liver, and in human lymphocyte and mouse L cell metaphase chromosomes in situ. By studying the rate of digestion of chromatin DNA by DNase, we have previously shown that DNA in extended chromatin is more sensitive to DNase digestion than that in condensed chromatin. In the current investigation, we have examined whether this differential sensitivity of the chromatin fractions to DNase is due to differences in protein binding to DNA or differences in the degree of chromatin condensation. By “decondensing” the condensed chromatin and comparing its rate of digestion to that of untreated condensed and extended chromatin, it was found that differences in the degree of binding of proteins to DNA rather than the degree of condensation of the chromatin primarily determines the sensitivity of each fraction to DNase. Extraction of the various classes of chromosomal proteins, followed by DNase digestion of the residual chromatin revealed that both the histone and non-histone proteins protect the DNA in the chromatin fractions from DNase attack; however, the more tightly associated non-histones appear to be specifically responsible for the differential sensitivity of the chromatin fractions to DNase digestion. These non-histones may be more tightly associated with the DNA in condensed than in extended chromatin, thereby protecting the DNA in condensed chromatin against DNase attack to a greater extent than that in extended chromatin. When metaphase chromosomes were briefly digested with DNase in situ and subsequently stained with Feulgen reagent, incontrovertible C-banding and some G-banding was obtained. This DNaseinduced banding demonstrates that the DNA in C-band and possibly G-band regions is less accessible to DNase than that in the interband regions, and our biochemical data suggest that this differential accessibility is caused by differential DNA-protein binding such that the non-histones are more tightly coupled to the DNA in the G- and C-band regions than they are in the interbands. Differences in the binding of non-histones to DNA in different segments of the metaphase chromosome may be involved in the mechanism of G- and C-banding.     

Distribution of estrogen receptors in subfractions of hen oviduct chromatin.

Hen oviduct chromatin was digested with DNase II and separated into two fractions. The MgCl2 insoluble chromatin fraction (43% of the total DNA) was enriched in nucleosome-like particles, which sedimented at 11 S and contained 185 base pairs of DNA. The MgCl2 soluble chromatin fraction (5% of the total DNA) was characterized by 5 S and 14 S peaks in sucrose gradients; Estrogen receptors in the chromatin fractions were labelled with [3H] estradiol using the steroid exchange assay. The concentration of receptors in the MgCl2 soluble chromatin was 4;5 times higher than that in the MgCl2 insoluble chromatinmin sucrose gradient analysis the 11 S particles displayed a negligible specific radioactivity suggesting that estrogen receptors mainly bind to extranucleosomal chromatin.     

Ionic requirements and subcellular localization of tubulin tyrosinolation in human polymorphonuclear leukocytes

We previously reported a specific stimulation of polymorphonuclear leukocyte (PMN) tubulin tyrosinolation as induced by the peptide chemoattractant N-formyl-methionyl-leucyl-phenylalanine (fmet-leu-phe) and the Ca2+ ionophore A23187 that is coupled to the NADPH oxidase-mediated stimulation of the PMN respiratory burst. The present study demonstrates that the presence of extracellular Ca2+ is necessary for fmet-leu-phe- and A23187-induced stimulation of PMN tubulin tyrosinolation, as indicated by the complete inhibition of the response by the addition of 1 mM EGTA to the extracellular medium. Methoxyverapamil (10(-5) M), a putative calcium channel blocker, completely inhibited the fmet-leu-phe-induced stimulation of tubulin tyrosinolation in PMN, but did not inhibit the A23187-induced response. Moreover, the calmodulin-binding drugs, trifluoperazine, fluphenazine, or chlorpromazine, at concentrations of 1 to 10 microM, caused significant inhibition of fmet-leu-phe- or A23187-induced stimulation of tubulin tyrosinolation. In related studies, enzymatic [14C]-tyrosinolation in isolated subcellular fractions of PMN revealed the presence of native tubulin in PMN fractions that were enriched in plasma membranes, the specific granules, or the azurophil granules. Most interestingly, tubulin tyrosine ligase (ligase), primarily a cytoplasmic enzyme, was detected in association with the PMN azurophil granule-rich fraction. Immunoautoradiography with the alpha-tubulin antibody YL 1/2 of isolated PMN subcellular fractions demonstrated a preferential stimulation of tyrosinolation of tubulin associated with the plasma membrane-rich fraction of fmet-leu-phe-stimulated cells. A significant stimulation was also observed in the cytoplasmic tubulin fraction. Consistent with the findings of in vitro tyrosinolation studies with PMN subcellular fractions, tyrosinolated tubulin was detected in the azurophil granule-enriched fractions isolated from both resting and fmet-leu-phe-stimulated cells. The antibody YL 1/2, which reacts with tyrosinolated alpha-tubulin and not with the detyrosinolated form, showed significant cross-reaction with several nontubulin PMN proteins.     

Distribution of Calretinin, Calbindin D28k, and Parvalbumin in Subcellular Fractions of Rat Cerebellum: Effects of Calcium

Abstract: The distribution of calretinin, calbindin D28k, and parvalbumin was examined in subcellular fractions prepared from rat cerebellum and analyzed by immunoblot. Calretinin was also quantified by radioimmunoassay. As expected, all three soluble, EF-hand calcium-binding proteins were predominantly localized in the cytosolic fraction. Calretinin and calbindin D28k were also detected in membrane fractions. Calretinin was more abundant in synaptic membrane than in microsomal fractions. The cerebellar microsomal fraction contained the greatest concentration of membrane-associated calbindin D28k. The association of calretinin and calbindin D28k with membrane fractions was decreased in samples prepared or incubated in low calcium. Quantification of calretinin in subcellular fractions of rat cerebellum revealed a greater amount of calretinin in cytosolic fractions prepared or incubated in low calcium and reduced amounts of calretinin in all membrane fractions incubated in low calcium with the exception of the mitochondrial fraction. These results imply that calretinin and calbindin D28k might have physiological target molecules that are associated with, or are components of, brain membranes.     

Interaction of extracellular vesicles of Bacteroides gingivalis W50 with human polymorphonuclear leucocytes

The effects of B. gingivalis W50 extracellular vesicles (ECV) on neutrophil chemotaxis and viability were assessed and compared with those of whole cells and the extracellular non-dialysable soluble protein (EP) fraction. None of the fractions tested, including soluble fractions derived from cells and ECV by sonication, induced neutrophil chemotaxis. Only ECV and cells inhibited f-MLP-stimulated chemotaxis. ECV and cells were cytotoxic towards neutrophils. The cytotoxic response was time dependent. The soluble EP fraction did not influence cell viability.     

Mitogenic and adjuvant activities of polysaccharides from the cellular slime mold,Dictyostelium discoideum NC-4

Cell surface and extracellular polysaccharide fractions obtained from Dictyostelium discoideum NC-4 cultured in bacteria-free medium showed strong B-cell mitogenic activities. Upon periodate treatment of the extracellular polysaccharide fraction this activity completely disappeared. The extracellular polysaccharide fraction could also enhance the antibody response in vitro against sheep red blood cells.     

ENZYME ACTIVITIES IN CHICK EMBRYONIC CARTILAGE : Their Subcellular Distribution in Isolated Chondrocytes

Some characteristic enzymatic activities were determined in chick embryonic cartilage and compared with the analogous activities in bone and liver. Chondrocytes were isolated, broken by sonication, and subjected to subcellular fractionation to yield a nuclear pellet, the mitochondrial, lysosomal, and microsomal fractions, and the high speed supernatant solution. It was established that these fractions are characterized by enzymatic activities usually associated with similar fractions in other tissues, but with some quantitative differences. Lysozyme, a particulate-associated enzyme in other tissues, was not detected in any subcellular fraction even by the sensitive technique of microzone electrophoresis and is therefore considered to be primarily extracellular in cartilage.